Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Anti-atherogenic effects of CD36-targeted epigallocatechin gallate-loaded nanoparticles

doi: 10.1016/j.jconrel.2019.04.018

Figure Lengend Snippet: The binding and uptake of NBD-labeled Vnano and L-Vnano by mouse peritoneal macrophages via their CD36 receptor. A, Mouse peritoneal macrophages were treated with non-targeted Vnano or targeted L-Vnano at 37°C; B, Mouse peritoneal macrophages were treated with Vnano or L-Vnano at 4°C; C, Mouse peritoneal macrophages were treated with red RPE-labeled anti-mouse CD36 antibody (λ of excitation is 496 nm, λ of emission is 578 nm) in combination with Vnano or L-Vnano at 4°C; D, Mouse peritoneal macrophages were transfected without CD36 siRNA (control) or with scramble siRNA (negative control) or with CD36 siRNA (CD36 knockdown). CD36 siRNA transfection decreased CD36 protein expression in mouse peritoneal macrophages; E, The binding and uptake of L-Vnano by control, CD36 negative control or CD36 knockdown mouse peritoneal macrophages. Bar length was 200 μm. NBD-labeled Vnano and L-Vnano were green (λ of excitation is 460 nm; λ of emission is 535 nm). Cell nuclei were stained by DAPI (λ of excitation is 358 nm, λ of emission is 461 nm) (blue color). Images were representatives of three independent experiments.

Article Snippet: Moreover, for competitive binding assay with mouse CD36 antibody, mouse peritoneal macrophages were treated with RPE-labeled anti-CD36 antibody (MCA2748PE, AbD Serotec, Raleigh, NC, USA) in combination with NBD-labeled Vnano or L-Vnano at 4°C for 2 hours.

Techniques: Binding Assay, Labeling, Transfection, Negative Control, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36 is expressed in the olfactory epithelium. (A) Quantitative real-time PCR showing expression of CD36 in adult mice compared to two olfactory receptors (mOR-EG and Olfr124). Data are the mean of five mice ( n = 5) and three technical replicates each. Significance was calculated using two sample t -test. Error bars represent SEM ( * p ≤ 0.05). (B) Western blot analysis of whole olfactory epithelium preparation, membrane preparation and supernatant of membrane preparation of an adult mouse showing CD36 protein expression. (C) Confocal image of a cryosection (P8) immunostained for CD36 (green) and the mature olfactory neuron marker OMP (red). CD36 is present in soma, dendrite and knob. (D) Confocal image (maximum projection) of an adult vomeronasal organ cryosection immunostained for OMP (red) and CD36 (green) showing densely packed vomeronasal sensory neurons but no CD36 presence in these neurons. (E) Confocal image of a cryosection (P8) immunostained for CD36 (green) and the mature olfactory neuron marker OMP (red) in CD36 −/− mice, showing absence of labeling. (F,G) Single channels of the picture shown in (E) . (H) RNA in situ hybridization of wild-type P8 cryosection showing CD36 mRNA expression in OSNs. (I) RNA in situ hybridization of cryosection from CD36 −/− mouse showing the absence of CD36 mRNA. Scale bars: 5 μm (C) , 20 μm (D) , 20 μm (E) , 50 μm (H) , 50 μm (I) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Marker, Labeling, RNA In Situ Hybridization

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36 distribution in the olfactory epithelium. (A) Widefield microscopy image of a cryosection (P8) immunostained for CD36. CD36 expressing neurons are scattered along the olfactory epithelium. (nc, nasal cavity; s, septum; t, turbinate; ob, olfactory bulb). (B) Magnification of the boxed area shown in (A) . Immunostaining for CD36 shows expression in a subset of olfactory neurons. (C) Stitching of fluorescence stereo microscopy images of a whole-mount turbinate preparation immunostained for CD36 (P8). Olfactory knobs of CD36 expressing neurons are detected as small dots all along the surface of the epithelium lining the turbinates Scale bar: 500 μm (A) , 50 μm (B) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Microscopy, Expressing, Immunostaining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36-positive glomeruli in the olfactory bulb. (A) Fluorescence stereo microscopy image (maximum projection) of an olfactory bulb preparation (P9) immunostained for CD36. Ventral view onto the right bulb. Medial side: top; lateral side: bottom. (B) Confocal image (maximum projection) of a vibratome section immunostained for CD36 demonstrating CD36 staining in glomeruli and nerve fibers (P9). (C–F) Fluorescence stereo microscopy images of P8 vibratome sections from rostral to caudal regions of the olfactory bulb immunostained for CD36. Glomeruli are located in ventromedial OB regions. Dotted line encircles the OB in the first image. (d, dorsal; l, lateral; v, ventral; m, medial, consistent for C–F ). Scale bars: 100 μm (B) , 200 μm (C–F) , 500 μm (A) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Fluorescence, Microscopy, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36 neurons express olfactory signal transduction proteins. (A–D) Confocal images of dissociated olfactory sensory neurons from P8 animals. Immunostainings show co-expression of CD36 (green) and Gα olf (A) , ACIII (B) , CNGA2 (C) , and ANO2 (D) (red). (E) Normalized CD36 read counts (Trimmed Mean of M -values (TMM) method) for both cell fractions CD36-depleted (CD36-dep) and CD36-enriched (CD36-enr) used for transcriptome sequencing. Diagram illustrates the enhancement of CD36 (3.4 fold) in the CD36-enriched fraction (257.5) compared to the CD36-depleted fraction (75.7). (F) Quantitative real-time PCR results for Gα olf , ACIII, CNGA2, and ANO2 comparing CD36-enriched and CD36-depleted cell fraction from magnetic CD36 cell sorting in P8 animals. Data was normalized to OMP. Scale bars: 5 μm (A–D) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Transduction, Expressing, Sequencing, Real-time Polymerase Chain Reaction, FACS

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36-positive neurons express olfactory receptors. (A) Western blot analysis of olfactory epithelium (1), membrane preparation (2), and supernatant (3) of three P60 mice showing panOR specificity to olfactory epithelium. Adult liver (4) was used as negative control; absence of GAPDH shows successful membrane preparation; mOR-EG demonstrates an olfactory receptor-specific band of same size like the panOR band. (B) Confocal image (maximum projection) of a cryosection immunostained with the panOR antibody (P8). Scale bar: 20 μm. (C–E) Confocal images (maximum projections) of a P8 cryosection immunostained for CD36 (green) and panOR (red). (F–H) Confocal images of P8 cryosections immunostained for CD36 (green) and specific olfactory receptors Olfr726 (F) , Olfr71 (G) , and mOR-EG (H) (red). Scale bars: 20 μm (B) , 10 μm (C–H) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Western Blot, Negative Control

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36 is localized in olfactory cilia . (A) Confocal image of a en face preparation immunostained for CD36 showing CD36 localization in olfactory cilia (P8). (B) Higher magnification of the boxed area in (A) . CD36 staining is prominent around the knob and along olfactory cilia. (C) Confocal image of an en face preparation immunostained for CD36 (green) and mOR-EG (red) showing differences in cilia length in both differently stained cell types, both proteins showed no co-expression. (D) Olfactory cilia counting: Confocal image of a top view preparation immunostained for CD36. Cilia were marked as demonstrated using a freehand tool from ImageJ. (E) Olfactory cilia counting: Confocal image of the same en face preparation shown in (C) , co-immunostained for mOR-EG. (F) Total number of cilia per knob for mOR-EG-positive and CD36-positive neurons. CD36-positive cells possess half as much cilia compared to mOR-EG expressing cells. Data are shown as mean ± SEM of 55 knobs in 3 individual preparations. Significance was calculated using two sample t -test ( * p ≤ 0.05). (G) Number of cilia with different length of mOR-EG-positive and CD36-positive neurons. CD36-positive cilia are shorter compared to mOR-EG-positive cilia. Data are shown as mean ± SEM of 55 knobs in 3 individual preparations. Significance was calculated using Mann–Whitney- U -Test ( * p < 0.01).

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: STED microscopy revealed receptor like localization of CD36 in olfactory cilia. (A,B) Shown are STED images of en face preparations immunostained for mOR-EG and CD36 (P8). (C,D) Higher magnifications of the cilia shown in (A,B) , respectively. The olfactory receptor mOR-EG and CD36 show a similar staining pattern. (E,F) High magnification pictures showing localization of CD36 and mOR-EG in discrete spots (examples are indicated by arrowheads). (G,H) STED images of dissociated adult neurons immunostained for acetylated tubulin (ac. tub). Scale bars: 1 μm (A–D,G,H) , 500 nm (E,F) . * Marks the dendritic knob (C,D,H) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Microscopy, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: Olfactory system of CD36 knockout mice. (A,B) Widefield fluorescence microscopy images of P8 wild type (A) and CD36 −/− (B) cryosections immunostained for the mature olfactory neuron marker OMP. Olfactory bulb and turbinates show no morphological abnormalities. (C,D) Head preparation of wild type (C) and CD36 −/− (D) P90 animals showing no differences in OB and OE size and shape. (E) Confocal image (maximum projection) of a P8 CD36 −/− cryosection immunostained for OMP (red), CD36 (green) and counterstained with TO-PRO (blue) to visualize cell nuclei. CD36 protein is absent in the olfactory epithelium. (F) Confocal image of a P14 CD36 −/− en face preparation immunostained for the olfactory receptor mOR-EG. Immunostaining shows normal mOR-EG localization to olfactory knobs and cilia and unaltered ciliary length. (G–N) Confocal images (maximum projections) of wild type (G–J) and CD36 −/− (K–N) cryosections immunostained for classical olfactory transduction proteins Gα olf , ACIII, CNGA2, and ANO2 (green) and counterstained with TO-PRO (blue). Signal cascade proteins are mainly restricted to the ciliary layer in both wild type and CD36 −/− P8 animals and no abnormal protein distributions are observed. Scale bars: 20 μm (E,F) , 50 μm (G–N) , 500 μm (A,B) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Knock-Out, Fluorescence, Microscopy, Marker, Immunostaining, Transduction

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: Functional investigation of CD36 knockout mice. (A,B) Representative traces of local field potentials (electro-olfactogram, EOG) generated in the main olfactory epithelium of wild type (A) and CD36 −/− (B) mice upon stimulation with a mixture of 100 odorants (Henkel 100). (C) Mean EOG responses to the odor mixture Henkel 100 from the main olfactory epithelium of wild type and CD36 −/− mice showing no significant differences. CD36 −/− mice possess normal ability to detect odorants. Data are shown as mean ± SEM of n = 6–8 mice at postnatal day 14 (3 individual recordings each). Significance was calculated using two sample t -test. Data are shown as mean ± SEM. (D,E) Representative traces (relative intensity vs. time) illustrating odor-, OA- and K + -dependent Ca 2+ elevations in olfactory neurons from a wild type (black) and a CD36 −/− mouse (red). Horizontal bars indicate stimulus application. OA concentrations as indicated. (F,G) Dose-dependent Ca 2+ elevations recorded from fluo-4/AM loaded OSNs in acute tissue sections from wild type mice. (F) Overlay of average Ca 2+ signals (gray curve; 11 randomly selected neurons) and an original trace from a single OSN (black) in response to increasing oleic acid (OA) concentrations (0.02, 0.2, and 20 μM). Relative fluorescence intensity is plotted as a function of time. Horizontal bars indicate duration of stimulus application. (G) Bar graph illustrating the relative proportion of OA-sensitive neurons as a function of stimulus concentration [ n = 27 (2 nM); n = 11 (20 nM); n = 11 (200 nM)]. Data are shown as mean ± SEM. (H–J) Bar charts comparing different response characteristics as a function of genotype [wild type (black) vs. CD36 −/− (gray)] and stimulus (odor-mix vs. OA). Signal parameters in response to odor (10 μM each) and OA (2 nM) are normalized to depolarization-dependent Ca 2+ signals (K + ; n = 11 randomly selected neurons). Maximum response amplitude [Ampmax (H)], rising speed [time-to-peak (I) and half-width; full duration at half maximum, FDHM (J) ] are plotted. Asterisks denote statistical significance (unpaired, two-sided t -test; * p ≤ 0.05; ** p ≤ 0.01; age P6–P9). Data are shown as mean ± SEM. (K) Bar diagram illustrating the portion of neurons responding to the odor-mix and to oleic acid, respectively, for wild type (black) and CD36 −/− (gray) animals. Cell count was normalized to K + -sensitive neurons in each experiment [wild type: n = 40 (odor-mix), 27 (OA, 2 nM), 37 (OA, 20 μM); CD36 −/− : n = 26 (odor-mix), 40 (OA, 2 nM and 20 μM)]. Similar portions of neurons responded to the odor mix. By contrast, the number of OA sensing neurons was significantly reduced in CD36 −/− mice. Significance was calculated using unpaired, two-sided t -tests ( ** p ≤ 0.01; *** p < 0.005; age P6–P9). Data are shown as mean ± SEM.

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Functional Assay, Knock-Out, Generated, Fluorescence, Concentration Assay, Cell Counting

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: CD36 localization is regulated. (A–F) Confocal images of en face olfactory epithelium preparations for age stages E18-P90 immunostained for CD36 (green) and mOR-EG (red) showing localization changes of CD36 in olfactory cilia. mOR-EG immunostainings validate successful sample preparation and show clear ciliary localization at all age stages. CD36 ciliary localization is restricted to P8 (C) and P14 (D) . Before, it is found in single olfactory knobs, but barely in cilia (E18: A ; P1: B ) and later, ciliary localization decreases (P21: E ) and is absent in adult animals (P90: F ). (G) Confocal image (maximum projection) of a P90 cryosection immunostained for CD36 (green) and OMP (red). CD36 is still localized in OSN soma and denrite in adult animals. Additionally, CD36 is expressed in the microvilli layer of sustentacular cells. (H) Cell counting of CD36 and OMP expressing neurons in a stretch of 600 μm epithelium lining the septum. Cell numbers for CD36 were normalized to OMP expressing cells. If possible, both sides of every septum were counted ( n = 2) to get a total number of 7–10. Significance was calculated in comparison to P8 using two sample t -test. Error bars represent SEM ( * p ≤ 0.05). Scale bars, 10 μm (A–F) .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Sample Prep, Cell Counting, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: CD36 is involved in oleic acid detection by the murine olfactory system

doi: 10.3389/fncel.2015.00366

Figure Lengend Snippet: Number of CD36 expressing cells in whole slices of age stages E18-P180 .

Article Snippet: Primary antibodies: rat anti-CD36 (AbD Serotec, MCA2748, MCA2748A488, dilution 1.200); goat anti-CNGA2 (Santa Cruz, M-20, dilution 1:200); rabbit anti-ACIII (Santa Cruz, C-20, 1:200); rabbit anti-ANO2, dilution 1:100 (Rasche et al., ), rabbit anti-mOR-EG, dilution 1:200 (Baumgart et al., ); rabbit anti-Gα olf (Santa Cruz, C18, dilution 1:200), goat anti-OMP (Wako, 019-22291, dilution 1:1000).

Techniques: Expressing